CLASSIFICATION OF Department of Math and Sciences

COLLEGE OF ARTS AND SCIENCES

MICROORGANISMS
Prepared by: Microbiology and Parasitology
DIVINE GRACE S. BATENGA, MSc., LPT
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CLASSIFICATION OF
MICROORGANISMS

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 Launched in 2001

ALL SPECIES
INVENTORY Purpose: to identify and record every species of life
on Earth in the next 25 years.

It is estimated that the number of living species

rangers from 10 to 100 million. 1.7 million identified.

Similarities:
All organisms are composed Store their genetic
of cells surrounded by a Use ATP for energy.
plasma membrane. information in DNA.

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 • Putting organisms into
TAXONOMY categories or taxa, to show
degrees of similarities
among organisms.

SYTEMATICS/ • The study of evolutionary

PHYLOGENY history of organisms.

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CLASSIFICATION OF ORGANISMS
qARISTOTLE – categorized organisms in two ways, plants and
animals.
qCAROLUS LINNAEUS – introduced a format system of
classification with two kingdoms – Plantae and Animalia
qCARL VON NÄGELI – proposed that bacteria and fungi be placed
in the plant kingdom.
qERNST HAECKEL – proposed the kingdom Protista to include
bacteria, protozoa, algae, Fungi.
qROBERT G. E. MURRAY – proposed the Kingdom Prokaryotae.

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CLASSIFICATION OF ORGANISMS

The Five-Kingdom System

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THE THREE DOMAINS
§ The discovery of three cell types was based on the observations that ribosomes are
not the same in all cells.
§ Comparing the sequences of nucleotides in ribosomal RNA from different kinds of
cells shows that there are three distinctly different cell groups.

EUKARYA BACTERIA ARCHEA

• Animals • Pathogenic prokaryotes • Prokaryotes that do not
• Plants • Non-pathogenic have peptidoglycan in
• Fungi prokaryotes their cell walls and carry
• Photoautotrophic out unusual metabolic
prokaryotes process.
• Often live in extreme
environments.
ü Methanogens
ü Extreme halophiles
ü Hyperthermophiles

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SCIENTIFIC A scientific name is usually
followed by the author’s name

NOMENCLATURE in an abbreviated form;

underlined if handwritten and
italicized if typewritten

vBINOMIAL NOMENCLATURE – organisms are given two names

§ Genus – always capitalize and is always a noun ü Scientific name
§ Specific epithet (species) – lowercase and is usually an adjective ü Latin language

a) Musca domestica Lin.

b b) Musca domestica Lin.
c) Musca domestica Lin.
d) Musca Domestica Lin.

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THE TAXONOMIC
HIERARCHY
Related kingdoms

Phyla that related to each other

Related classes

Similar orders

Similar families

Related genera

Related species
Closely related organisms that
can interbreed
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Domestic dog

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CLASSIFICATION OF
PROKARYOTES
vThe taxonomic classification for prokaryotes is found in Bergey’s Manual of
Systematic Bateriology 2nd Edition. (Appendix E).
DOMAIN DOMAIN
BACTERIA ARCHEA

Phyla
Orders
Families
Genera
Species
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 Prokaryotic Species
vA population of cells with similar characteristics.
§ Members of a bacterial species are nearly indistinguishable
from each other but are distinguishable from members of
other species usually on the basis of several features.
§ CULTURE – bacteria grown in media
§ MIXED CULTURE – more than one organism
§ PURE CULTURE – often a clone, a population of cells
derived from a single parent cell.
§ STRAIN – pure cultures of the same species that are not identical;
subtype of microorganism

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https://www.youtube.com/watch?v=gwPZhsyMI9M – spread plate
https://www.youtube.com/watch?v=lCVl3l7GmRI – pour plate
https://www.youtube.com/watch?v=bm99zrq3ijo – streak plate

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 CLASSIFICATION OF
EUKARYOTES
KINGDOM FUNGI
vIncludes unicellular yeasts, multicellular
molds, and macroscopic species such as
mushrooms.
vAbsorbs dissolved organic matter to
obtain raw materials for vital functions.
vFungi develop from spores or from
fragments of hyphae.
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 CLASSIFICATION OF
EUKARYOTES
KINGDOM PLANTAE
vMosses, ferns, conifers, and flowering
plants.
vAll members of this kingdom are
multicellular.
vThey use photosynthesis, process that
converts carbon dioxide and water into
organic molecules used by the cell.
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 CLASSIFICATION OF
EUKARYOTES
KINGDOM ANIMALIA
vSponges, worms, insects, and animals
with backbones (vertebrates).
vObtain nutrients and energy by
ingesting organic matter through a
mouth of some kind.

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 CLASSIFICATION OF VIRUSES
vViruses aren’t classified as part of any
of the three domains.
vThey aren’t composed of cells, and
they use the anabolic machinery within
living host cells to multiply.
vViral genome can direct biosynthesis
inside a host cell, and some viral
genomes can be incorporated into the
host cell.

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 CLASSIFICATION OF VIRUSES
vThe ecological niche of a virus is its
specific host cell.
vViruses may be more closely related to
their hosts than to other viruses.
vViruses are obligatory intracellular
parasites.
vViral genes carried in the genomes of
other organisms provide a record of viral
evolution.
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Viral Species
vA population of viruses
with similar characteristics
(morphology, genes,
enzymes) that occupies a
particular ecological niche.

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Hypotheses on the origin of viruses
1) They arose from independently
replicating strands of nucleic acids
(plasmids).
2) They developed from degenerative
cells that, through many
generations, gradually lost the
ability to survive independently
but could survive when associated
with another cell.
3) They coevolved with host cells.

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METHODS OF
CLASSIFYING AND
IDENTIFYING
MICROORGANISM

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CLASSIFICATION
• Placing organisms in groups of related
species.
• Lists of characteristics of known
organisms.
IDENTIFICATION
• Matching characteristics of an “unknown”
to lists of known organisms.

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MORPHOLOGICAL
CHARACTERISTICS
vHigher organisms are frequently classified according to
observed anatomical detail.
vMany microorganisms look too similar to
be classified by their structures alone.
§ Organisms that might differ in metabolic or
physiological properties may look alike under a
microscope.

üMorphological characteristics are still

useful in identifying bacteria to know the
differences in structures such as
endospores or flagella.

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DIFFERENTIAL STAINING
vOne of the first step in identifying bacteria is
differential staining.
§ Gram staining
§ Acid-fast staining

vRecall: These stains are based on the chemical

composition of cell walls and not useful in
identifying wall-less bacteria and unusual walls of
archaea.
vMicroscopic examination of a Gram stain or an
acid-fast stain is used to obtain information
quickly in the clinical environment.
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BIOCHEMICAL TEST
vEnzymatic activities are widely used to differentiate
bacteria.
vClosely related bacteria can usually be separated into
distinct species by subjecting them to biochemical tests.
vBiochemical test can provide insight into species’ niche in
the ecosystem.
§A bacterium that can fix nitrogen gas or oxidize elemental sulfur will provide
important nutrients for plant and animals.

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Enteric gram-negative bacteria
§ Large heterogenous group of
microbes whose natural habitat is the qLACTOSE FERMENTING
intestinal tract of humans and other üEscherichia, Enterobacter, Citrobacter
animals.
qNON-LACTOSE FERMENTING
§ This family contains several üSalmonella and Shigella
pathogens that cause diarrheal illness.
§ All members of the family
Enterobacteriaceae are oxidase-
negative.
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AEROBIC AND ANAEROBIC

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The time needed to identify bacteria can be reduced
by the use of selective and differential media.
SELECTIVE MEDIA
REVIEW!!!
• Contains ingredients that suppress the
growth of competing organisms and
encourage the growth of desired ones.
DIFFERENTIAL MEDIA
• Contain compounds that allow groups
of microorganisms to be visually
distinguished by the appearance of the
colony or the surrounding media.

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 MacConkey Agar (Differential and
Selective)
ü Used for the isolation and differentiation of non-
fastidious gram-negative rods, particularly
members of the family Enterobacteriaceae and the
genus Pseudomonas.

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Brilliant Green Agar (Differential and
Selective Media)

ü It was first described as a selective isolation

medium for Salmonella species.
ü Brilliant Green Agar medium is
recommended as a primary plating medium
for isolation of Salmonella species.

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 Eosin Methylene Blue Agar
(Differential and Selective Media)

ü Eosin Methylene Blue (EMB) agar is a

differential microbiological medium, which
slightly inhibits the growth of Gram-positive
bacteria and provides a color indicator
distinguishing between organisms that ferment
lactose (e.g., E. coli) and those that do not (e.g.,
Salmonella, Shigella).

E. Coli - Blue-black bulls eye; may have

green metallic sheen
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 Cetrimide Agar (Selective Media)

ü Cetrimide Agar is a selective medium used

for the isolation and identification of
Pseudomonas aeruginosa from clinical and non-
clinical specimens.
ü Cetrimide is the selective agent and inhibits
most bacteria by acting as a detergent.

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Limitation of Biochemical Test

A limitation of
biochemical testing is that Unless a large number of
mutations and plasmid tests is used, an organism
acquisition can result in could be incorrectly
strains with different identified.
characteristics.

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 Rapid Identification Method
vManufactured for groups of
medically important bacteria.
vSuch tools are designed to
perform several biochemical
tests simultaneously and can
identify bacteria within 4 to 24
hours.

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 SEROLOGY
vThe science that studies serum and
immune responses that are evident in
serum.
vMicroorganisms are antigenic!
vAntibodies are proteins that circulate in
the blood and combine in a highly
specific way with the bacteria that
caused their production.
vANTISERUM – solutions of
antibodies; commercially available
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 Slide Agglutination Test
1) Samples of the unknown bacterium are placed
in a drop of saline of each of several slides.
2) A different known antiserum is added to each
sample.
3) The bacteria agglutinate (clump) when mixed
with antibodies that were produced in
response to that species or strain of
bacterium.
A positive test is indicated by the presence of
agglutination.

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 Enzyme-linked immunosorbent assay
(ELISA)
vWidely used because it is fast and can be
read by computer scanner.
1) Known antibodies are placed in the wells of a
microplate.
2) An unknown type of bacterium is added to each
well.
3) A reaction between the unknown antibodies and
the bacteria provides identification of the bacteria.

ELISA is used in AIDS testing to detect the

presence of antibodies against human
immunodeficiency virus (HIV)

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 Western Blotting
vA laboratory technique used to detect
a specific protein in a blood or tissue
sample.
§ The method involves using gel
electrophoresis to separate the sample's
proteins.
§ The separated proteins are transferred out
of the gel to the surface of a membrane.
HIV infection is confirmed by Western
blotting, and Lyme disease caused by
Borrelia burdorferi, is often diagnosed by
Western blot.

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 PHAGE TYPING
vLooks for similarities among bacteria.
vUseful in tracing the origin and course of a
disease outbreak.
vIs a test for determining to which phages
a bacterium is susceptible.
vBACTERIOPHAGES (phages) – are
bacterial viruses that cause lysis of the
bacterial cells they infect.
§ Highly specialized, they infect only members of a
particular species.
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Phage typing procedure
1) This procedure starts with a plate
totally covered with bacteria growing
on agar.
2) A drop of each different phage type to
be used in the test is then placed on
the bacteria.
Wherever the phages are able to infect
and lyse the bacterial cells, clearings in
the bacterial growth (plaques) appear.

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 Phage typing (clinical importance)
“Bacterial infection on a surgical wound”

= =
CONCLUSION
Bacteria isolated from surgical wound have the
same pattern of phage sensitivity as those
The surgeon or a nurse is the source of
isolated from the operating surgeon or surgical infection.
nurses. 45
DNA SEQUENCING
vTaxonomists can use an organism’s DNA base
composition to draw conclusion about relatedness.
vThis base composition is usually expressed as the
percentage of guanine plus cytosine (C+G). ü The percentage of DNA
bases that are GC pairs
vRecall: also tells us the
(G) guanine – (C) cytosine percentage that are AT
(A) adenine – (T) thymine Fixed property pairs.

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DNA Sequencing Interpretation
10%
diff
eren
50% 50% ce 60%
50 60
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40
50 40%
35 40
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%CG

%CG
25 30
20
15 20

10 10
5
0 0
Percentage in CG pairs Percentage in CG pairs
Organism 1 organism 2 Organism 1 organism 2

Closely related Not related

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 DNA FINGERPRINTING
vThe use of restriction enzymes enables
researchers to compare the base
sequences of different organisms.
vIn this technique, the DNA from two
microorganisms is treated with the
same restriction enzyme.
vRESTRICTION FRAGMENT – a
DNA fragment resulting from the
cutting of a DNA strand by a
restriction enzyme. DNA Fingerprints of seven different bacteria. Comparison
of the lanes shows that DNA samples in lanes 2 and 3; 4 and 5; and 1
and 6 are identical. 49
 Steps in DNA Fingerprinting
1) DNA EXTRACTION
Ø Cells are broken down to release DNA.
Ø If only a small amount of DNA is available, it can be
amplified using polymerase chain reaction (PCR).

2) CUTTING THE DNA

Ø The DNA is cut into fragments using restriction
enzymes.

3) GENERATION OF DNA FRAGMENTS

Ø PCR

4) SIZING AND SORTING THE DNA

Ø Fragments are separated on the basis of size using a
process called gel electrophoresis.
Ø An electrical current was then applied to the gel to
separate the fragments by size.
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DNA Fingerprinting (clinical
importance)
o In one hospital, patients
undergoing coronary bypass
surgery developed infections
caused by Gordonia bronchialis.
o A DNA sample from the
bacterium, and a bacterial samples
from surgeon and three (3) nurses
was obtained.
o The DNA profiles of the
bacterium, the surgeon and the
three (3) nurses as follows:

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The hospital was thus able to break the infection’s
chain of transmission by encouraging the Nurse 2
to use aseptic technique.
Bacterium Surgeon Nurse 1 Nurse 2 Nurse 3
Profile

Who causes the infection? 52

 Other application of DNA Fingerprinting

Forensics Paternity/Maternity Test

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 NUCLEIC ACID HYBRIDIZATION
Note:
vIf double stranded molecule of DNA
is subjected to heat, the
complementary strands will
separate as the hydrogen bonds
between the bases break.
vIf the single strands are cooled
slowly, they will reunite to form a
double-stranded molecule identical to
the original double strand.
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 NUCLEIC ACID HYBRIDIZATION
ü The procedure assumes that if two species are
similar or related, a major portion of their nucleic
acid sequences will also similar.
ü The procedure measures the ability of DNA
strands from one organism to hybridize (bind
through complementary base pairing) with the
DNA strands of another organism.
ü Similar hybridization reactions can occur between
any single stranded nucleic acid chain: DNA-
DNA, RNA-RNA, DNA-RNA.
Hyrbridization of 70% or more indicates the
two organisms belong to the same species.
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 Nucleic Acid Amplification Tests
(NAATs)
vWhen a microorganisms cannot be
cultured by conventional methods, the
causative agent of an infectious disease
might not be recognized.
vNAATs – can be used to increase the
amount of microbial DNA to levels that
can be tested by gel electrophoresis.
vNAATs use PCR
üReverse-transcription PCR
üReal-time PCR PCR
Polymerase Chain Reaction
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NAATs (Result)
üIf a PRIMER for a specific microorganism is used, the presence of amplified
DNA indicates that the microorganism is present.

16S rRNA PRIMER

is used as a tool to
identify bacteria at
the species level.

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 NAATs clinical application
§ In 1992, researchers used PCR to determine the
causative agent of Whipple’s disease, which was
previously an unknown bacterium now named
Tropheryma whipplei.
§ Whipple’s disease was first described in 1907 by
George Whipple as a gastrointestinal and nervous
system disorder caused by an unknown bacillus.
§ No one has been able to culture the bacterium to
identify it.
§ PCR provides the only reliable methods of
diagnosing and treating the disease.
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NAATs clinical application
§ PCR is used to diagnosis
Zika virus in pregnant
women who may have been
exposed to the virus.
§ PCR is used to identify the
source of rabies viruses as
well.

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NAATs clinical application
§ In 2013, public health
scientists used real-time PCR
to identify a new strain of
H7N9 influenza virus.

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 NAATs clinical application
§ Real-time reverse transcription polymerase
chain reaction (rRT-PCR) test for the
qualitative detection of nucleic acid from SARS-
CoV-2.
§ Coronaviruses, including the pneumonia-causing
novel coronavirus currently known as SARS-
CoV-2, are positive-sense RNA viruses.
§ Reverse transcription PCR, or RT-PCR, allows
the use of RNA as a template.
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 Southern Blotting
vA method used in molecular biology
for detection of a specific DNA
sequence in DNA samples.
vSouthern blotting combines transfer
of electrophoresis-separated DNA
fragments to a filter membrane and
subsequent fragment detection
by probe hybridization.

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 DNA Chips
(Microarray)
vCan quickly detect a pathogen in a host or the
environment by identifying a gene that is
unique to that pathogen.
vThe DNA chip is composed of DNA probes.
vDNA probes are stretches of single-stranded
DNA used to detect the presence of
complementary nucleic acid sequences (target
sequences) by hybridization.
§ A sample containing DNA from an unknown
organism is labeled with fluorescent dye and
added to the chip.
§ Hybridization between the probe DNA and DNA
in the sample is detected by florescence.
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 Ribotyping and Ribosomal RNA
Sequencing
vRibotyping – currently being used to determine the
phylogenetic relationships among organisms.
vAdvantages:
1. All cells contain ribosomes.
2. RNA genes have undergone few changes overtime. (all members of a
domain, phylum, and genus have the same “signature” sequences in
their rRNA)
3. Cells don’t have to be cultured in the laboratory.

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 Ribotyping and Ribosomal RNA
Sequencing
vMethods:
§ DNA can be amplified by PCR using rRNA primer for specific signature sequences.
§ Amplified fragments are subsequently cut with restriction enzymes and separated by
electrophoresis.
§ The resulting band patterns can then be compared.
vImportance:
§rRNA genes in the amplified fragments can be sequenced to determine
evolutionary relationships between organisms.
§Useful in classifying a newly discovered type of organisms present in one
environment.

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 Fluorescent In Situ Hybridization
(FISH)
vFluorescent dye-labeled RNA or DNA probes are used to stain microorganisms
in place.
vMethods:
§ Cells are treated so the probe enters the cells and reacts with the target DNA in the cell (in
situ).
vUsefulness:
§ Used to determine the identity, abundance, and relative activity of microorganisms in
an environment.
§ Can be used to detect bacteria that have not yet been cultured.
§ As probes developed, FISH can be used to detect bacteria in drinking water or bacteria
in a patient without the normal 24-hour or longer wait required to culture the bacteria.

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Fluorescent In Situ Hybridization
(FISH)

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We need both new and old
techniques to properly study
microbiome.

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PUTTING
CLASSIFICATION
METHODS
TOGETHER

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 Dichotomous Keys
vWidely used for identification.
vIdentification is based on successive
questions.
vEach question has two possible answers
(dichotomous = cut in two)
§ After answering one question, the investigator is
directed to another question until the organism is
identified.
vOften have little to do with phylogenetic
relationships.
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 Cladograms
vMaps that show evolutionary relationships
among organisms.
vClado – branch
vEach branch point is defined by a feature
shared by various species on that branch.
vMost microorganisms don’t leave fossils!
§ rRNA sequencing is primarily used to make
cladograms for microorganisms.
§ The small rRNA subunit used has 1500 bases.
§ Computer programs do the calculations.

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 CANVAS ACTIVITY…

§ As a hospice nurse, you are caring for a 75-year-

old patient undergoing chemotherapy for cancer
who recently developed pneumonia.
§ The patient is homebound, and none of his
visitors have been sick.
In the Clinic § While at the patient’s house to collect sputum
sample for the laboratory, you notice the man’s
dog also has a cough.
§ You end up swabbing the pet’s nose and sending
sample in, too.
§ The cultures for both man and dog grow
gram-negative, oxidase-positive, urease- and
H2S positive bacteria. WHAT IS THE
CAUSATIVE AGENT OF THESE
INFECTIONS?
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BACTERIAL ID
§ Organism: _____________
§ Verify your result by making a research about the identified
organism.

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End of Lecture…

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References:
§ Tortora GJ, Funke BR, Case CL. (2019). Microbiology: An
Introduction, 13th Edition. Pearson Education.

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